Deoxoartemisinin analogs, process for their preparation, and anticancer agent comprising them

ABSTRACT

The present invention relates to a new deoxoartemisinin dimer and trimer, which have excellent anticancer activity and lower toxicity and are stable to acids, to a new deoxoartemisinin monomer of intermediate thereof, to preparations thereof, and to anticancer agents comprising the deoxoartemisinin dimer or trimer.

TECHNICAL FIELD

The invention relates to deoxoartemisinin analogs, process for theirpreparation and anticancer agent comprising them. More particularly, thepresent invention relates to a new deoxoartemisinin dimer and trimer,which have excellent anticancer activity and lower toxicity and are acidstable, to a new deoxoartemisinin monomer of intermediate thereof, topreparations thereof, and to anticancer agents comprising thedeoxoartemisinin dimer or trimer.

BACKGROUND ART

Artemisinin (Qinghaosu)(I), a sesquiterpene lactone endoperoxide, is thefirst natural trioxane isolated from Artermisia annua, L.

The artemisinin is of special biological interest because of itsoutstanding antimalarial activity and outstanding activity againstpneumocystis carinii and toxoplasma gondii. The anti-humanimmunodeficiency virus (HIV) activity of artemisinin derivatives hasbeen also reported. Artemisinin has been subjected to a number ofreviews because of its novel structure and outstanding antimalarialactivity. Most first generation C-12 acetal type derivatives arehydrolytically unstable. Also, most semi-syntheses have involvedreplacing the C-12 acetal functionality in ether derivatives by lesshydrolytically prone functional groups. Recently, however, C-12non-acetal-type deoxoartemisinin (III) prepared from either artemisininof formula (I) or artemisinic acid of formula (II) has been reported toshow more antimalarial activity than that of artemisinin both in vitroand in vivo (see Jung, M.; Li, X.; Bustos, D. A.; ElSohly, H. N.;McChesney, J. D., A Short and Stereospecific Synthesis of(+)-Deoxoartemisinin and (−)-Deoxodesoxyartemisinin, Tetrahedron Lett.,1989, 30, 5973–5976 and Jung, M.; Li, X.; Bustos, D. A.; ElSohly, H. N.;McChesney, J. D.; Milhous, W. K., Synthesis and Antimalarial Activity of(+)-Deoxoartemisinin, J. Med. Chem., 1990, 33, 1516–1518).

Non-acetal-type analogs of deoxoartemisinin recently received attentionowing to their better bioavailability, such as acid stability, thanacetal-type analogs. Furthermore, evidence that analogs not possessingexo-oxygen at C-12 are less neurotoxic in animal studies than acetaltype artemisinin is also emerging and may thus lead to the futureabandonment of the currently clinically used acetal-type analogs (e.g.,arteether, artemether, artesunate and artelinic acid). After thepreparation of 12-n-butyldeoxoartemisinin as the first hydrolyticallystable non-acetal type analog containing a C—C bond at C-12 wasreported, a series of non-acetal-type derivatives including a few ofheteroaryl and unsaturated substituents a C-12 have been prepared (SeeJung, M.; Bustos, D. A.; ElSohly, H. N.; McChesney, J. D., A Concise andStereoselective Synthesis of (+)-12-n-Butyldeoxoartemisinin, Synlett,1990, 743–744 and Chorki, F.; Crousse, B.; Bonnet-Delpon, D.; Begue, J.P.; Brigaud, T.; Portella, C., C-10 Fluorinated Derivatives ofDihydroartemisinin: Difluoromethylene Ketones, Tetrahedron Lett., 2001,42, 1487–1489).

Although most studies have focused on antimalarial activities, a fewresearch groups have recently reported on cancer cell toxicity ofartemisinin and it related derivatives. (see Woerdenbag, H. J.; Moskal,T. A.; Pras, N.; Maringle, T. M.; ElFeraly, F. S.; Kampinga, H. H.;Konings, A. W. T., Cytotoxicity of Artemisinin-related Endoperoxides toEhrlich ascites Tumor cells, J. Nat. Prod., 1993, 56, 849 and Wu, J-M.;Shan F.; Wu, G-S.; Li, Y.; Ding, J.; Xiao, D.; Han, J-X.; Atassi, G;Leonce, S.; Caignard, D-H.; Renard, P., Synthesis and Cytotoxicity ofArtemisinin derivatives containing Cyanoarylmethyl group, Eur. J. Med.Chem., 2001, 36(5), 469–479). Because of their higher rate of celldivision, most cancer cells express a higher surface concentration oftransferrin receptors than normal cells and have high rates of ironintake. A unique structure bearing endoperoxide could be a trigger forthe generation of active oxygen radicals via hemolytic cleavage of theweak oxygen-peroxide bond accelerated by higher ferrous ionconcentration of cancer cell, which may mediate for the selective andpreferable damage to vital cellular structures of the relatively activecancer cells. Although some dimers of acetal type derivatives ofartemisinin have been prepared and show anticancer activities, yieldsare low and most of them possess either aromatic linkers or still acetaltypes at the C-12 position, which are neurotoxic, acid unstable, andshow low anticancer activities (see Galal, A. M.; Ahmad, M. S.;El-Feraly, F. S., Preparation and Characterization of a NewArtemisinin-Derived Dimer, J. Nat. Prod., 1996, 59, 917–920; Posner, G.H.; Ploypradith, P.; Parker, M. H.; O'Dowd, H.; Woo, S-H.; Northrop, J.;Krasavin, M.; Dolan, P.; Kensler, T. W.; Xie, S.; Shapiro, Antimalarial,Antiproliferative, and Antitumor Activities of Artemisinin-Derived,Chemically Robust, Trioxane Dimers., J. Med. Chem., 1999, 42, 4275–4280;and Ekthawatchai, S.; Kamchonwongpaisan, S.; Kongsaeree, P.;Tarnchompoo, B.; Thebtaranonth, Y; Yuthavong, Y, C-16 ArtemisininDerivatives and Their Antimalarial and Cytotoxic Activities: Synthesisof Artemisinin Monomers, Dimers, Trimers, and Tetramers by NucleophilicAdditions to Artemisitene, J. Med. Chem., 2001, 44, 4688–4695).

DISCLOSURE

Suprisingly, we found that deoxoartemisinin dimer and trimer hadexcellent anticancer activity, by preparing non-acetal-typedeoxoartemisinin dimer and trimer having no linker containing C—O bonds,aromatic or unsaturated groups at C-12 position and then testing theanticancer activity thereof.

Accordingly, it is an object of the present invention to provide adeoxoartemisinin dimer and trimer having excellent anticancer activity.

It is another object of the present invention to easily prepare thedeoxoartemisinin dimer and trimer in high yield.

It is another object of the present invention to provide anticanceragents comprising the deoxoartemisinin dimer and trimer.

It is another object of the present invention to provide adeoxoartemisinin monomer of intermediate for preparing thedeoxoartemisinin dimer and trimer in high yield.

It is another object of the present invention to easily prepare thedeoxoartemisinin monomer in high yield.

Deoxoartemisinin dimer and trimer of the present invention have thefollowing formula (IV):

wherein Y is —S—, —SO₂—,

The deoxoartemisinin dimer and trimer of the present invention areprepared from the deoxoartemisinin monomers of formula (V) and (VI),respectively, as shown in Schemes 1, 2 and 3 below.

The deoxoartemisinin dimer of the present invention having sulfide- orsulfonyl group-bearing linker is prepared by bis-nucleophilic couplingreaction of 12-bromoethyldeoxoartemisinin monomers (V), as shown inScheme 1 below. Particularly, the deoxoartemisinin dimer (IVa₁) isprepared by reacting 1 mole of sodium sulfide with 2 moles of12-bromoethyldeoxoartemisinin (V) and dimer (IVa₂) is prepared byoxidizing the dimer (IVa₁) with oxidizing agent such asmeta-chloroperbenzoic acid (m-CPBA). In a similar fashion, thedeoxoartemisinin dimer (IVb₁) is prepared by 1 mole of1,3-propanedithiol with 2 moles of 12-bromoethyldeoxoartemisinin (V) andthe dimer (IVb₂) is prepared by reacting the dimer (IVb₁) with anoxidizing agent such as m-CPBA.

-   Scheme 1

The deoxoartemisinin dimer of the present invention having amidegroups-containing linker is prepared from 12-aminoethyldeoxoartemisinin(VI), as shown in Scheme 2 below. Particularly, the deoxoartemisinindimer (IVc) is prepared by coupling 12-aminoethyldeoxoartemisinin (VI)with 12-carboxylethyldeoxoartemisinin (VII) in the presence of acatalyst such as1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC)/10-hydroxybenzotriazole(HOBt).On the other hand, the deoxoartemisinin dimer (IVd) is prepared bydirectly coupling 12-aminoethyldeoxoartemisinin (VI) with protectedglutarate in the presence of a catalyst such as EDC/HOBt, removing thebenzyl group of the ester, coupling the resultant product with12-aminoethyldeoxoartemisinin (VI) in the presence of a catalyst such asEDC/HOBt and then deprotecting the t-BOC protected group of the aminogroup of the resultant product.

-   Scheme 2

The deoxoartemisinin trimer (IVe) of the present invention is preparedby coupling 12-carboxylethyldeoxoartemisinin (VII) with L-glutamicdiethylester in the presence of a catalyst such as EDC/HOBt, hydrolyzingthe two ester groups of the product from the previous step and thendoubly coupling the product from the previous step with 2 moles of12-aminoethyldeoxoartemisinin (VI) in the presence of a catalyst such asEDC/HOBt, as shown in Scheme 3 below.

The novel deoxoartemisinin monomers (V and VI), which are intermediatesfor preparing the deoxoartemisinin dimer and trimer of the presentinvention, are prepared from 12-vinyldihydroartemisinyl alcohol (XIII),as shown in Scheme 4 below. A 12-vinyldihydroartemisinyl alcohol (XIII)can be synthesized from artemisinic acid (II) by a known synthetic route(see Jung et al., A Concise and Stereoselective Synthesis of(+)-12-n-Butyldeoxoartemisinin, Synlett, 1990, 743–744). Sinceartemisinin is much more expensive than artemisinic acid and a directintroduction of a C—C bond at C-12 of artemisinin may cause adestruction of the biologically essential endoperoxide, the artemisinicacid is used. Particularly, 12-bromoethyldeoxoartemisinin (V) isprepared by direct hydroborative oxidizing a terminal olefin of12-vinyldihydroartemisinyl alcohol (XIII), brominating the resultantproduct with CBr₄/PPh₃, photooxygenative cyclizing the resultant productby a known procedure (see Jung, M et al., supra). On the other hand,12-aminoethyldeoxoartemisinin (VI) is prepared by reacting12-bromoethyldeoxoartemisinin (V) with sodium azide and then reducingthe azide of the resultant product. In the preparation of12-bromoethyldeoxoartemisinin (V) and 12-aminoethyldeoxoartemisinin (VI)according to the above-mentioned procedures, 12β-epimer is obtainedexclusively.

The deoxoartemisinin dimer and trimer of the present invention is C-12non-acetal-type and have no linker containing C—O bonds, aromatic orunsaturated groups at C-12 position, and thus is less neurotoxic, acidstable and have a higher anticancer activity. Therefore, the dimer andtrimer of the present invention can be used as an effective anticanceragent for oral administration.

An anticancer agent containing the deoxoartemisinin dimer or trimer ofthe present invention as an effective constituent can be administeredorally (e.g., ingestion or inhalation) or parenterally (e.g.,intravenous injection, subcutaneous injection, percutaneous absorption,etc.) and can be prepared in the various form of tablets, capsules,granules, fine subtilae, powders, sublingual tablets, suppositories,ointments, injections, emulsions, suspensions, drug-treated syrups,etc., depending on its use. The above-mentioned various type ofanticancer agents are prepared by a known technique usingpharmaceutically acceptable conventional carrier such as excipient,binder, disintegrator, lubricant, antiseptic, antioxidant, isotonicagent, buffer, coating, sweeting, solubilizer, base, dispersion,stabilizer, colorant.

In the preparation of the agent, the content of the compounds of thepresent invention depends on the type of agents, but preferably rangesfrom 0.01 to 100% by weight.

The dosage of the anticancer agent of the present invention will varydepending on a variety of factors, such as, the kind of mammalianincluding human to be treated, the severity of the disease and thephysician's judgment. Typically, for oral administration, the anticanceragent of the present invention can be administered as an effectiveconstituent in the amount of 0.01 to 50 mg per kg body weight per dayand, for parenteral administration, in the amount of 0.01 to 10 mg perkg body weight per day.

The anticancer agent of the present invention can be administered at onetime or at several times per day and its dosage can be varied dependingon the severity of the disease and the physician's judgment.

The present invention is further described in the following examples.These examples illustrate the invention only and are not to be construedas limiting the scope of the present invention in any way.

EXAMPLES Example 1 Synthesis of 12-(2′-bromoethyl)deoxoartemisinin (V)(1) Synthesis of 12-(2′-hydroxyethyl)dihydroartemisinyl alcohol (XIV)

12-vinyldihydroartemisinyl alcohol (XII) (568 mg, 2.290 mmol), preparedby a known procedure (see Jung et al., A Concise and StereoselectiveSynthesis of (+)-12-n-Butyldeoxoartemisinin, Synlett, 1990, 743–744),was slowly added to THF solution containing 0.5M of9-borobicyclo[3.3.1]nonan (9BBN) (90.1 mL, 4.58 mmol) under nitrogenatmosphere and the resulting mixture was stirred at room temperature for30 minutes. Then, to this solution was added 30%—H₂O₂/3N—NaOH (1/1, 2mL) and the resulting solution was stirred at room temperature for 1hour. The reaction mixture was extracted with ether (40 mL×2) and washedwith saturated NaHCO₃ (20 mL) and brine (20 mL×2). The extract was driedover MgSO₄, concentrated in vacuo and purified by silica gel column(hexane/ethyl acetate=1/1 as eluent) to afford12-(2′-hydroxyethyl)dihyroartemisinyl alcohol (XIV) as a colorless oil(97% yield).

¹H-NMR (CDCl₃, 250 MHz) δ5.15(s, 1H, H-5), 4.14(d, 1H, J=9.8 Hz, H-12),3.89–3.80(m, 2H, H-2), 2.47(s, 1H), 1.94–1.72(m, 6H), 1.54(s, 3H,CH₃-15), 1.53–1.25(m, 9H), 0.84(d, 3H, J=7.3 Hz, CH₃-13), 0.87(d, 3H,J=6.6 Hz, CH₃-14).

¹³C-NMR(CDCl₃, 63 MHz) δ135.5, 120.9, 72.0, 62.6, 42.6, 42.5, 39.7,37.8, 37.6, 36.0, 28.1, 27.0, 26.4, 26.2, 24.1, 20.1, 10.4.

IR(neat) u_(max) 3435(OH), 2921, 1647, 1622, 1386, 1124, 1088, 1016 cm⁻¹MS(EI) m/z 266([M+]), 248([M+]—H₂O), 203([M+]—C₂H₅O).

(2) Synthesis of 12-(2′-bromoethyl)dihydroartemisinyl Alcohol (XV)

12-(2′-hydroxyethyl)dihydroartemisinyl alcohol (XIV) (399 mg, 1.503mmol) was dissolved in dry CH₂Cl₂ (20 mL) and triphenylphosphine (TPP)(393 mg, 1.503 mmol) was added. The solution was allowed to stir at 0°C. for 30 minutes. The reaction mixture was warmed to room temperature.To this reaction mixture was slowly added CBr₄ (498 mg, 1.503 mmol). Thereaction was allowed to stir at room temperature for 30 minutes and wasquenched with methanol (10 mL). The reaction mixture was extracted withethyl acetate (20 mL×3) and was washed with brine (20 mL×2). The extractwas dried over MgSO₄, concentrated in vacuo and purified by silica gelcolumn (hexane/ethyl acetate=5/2 as eluent) to afford12-(2′-bromoethyl)dihydroartemisinyl alcohol (XV) (470 mg, 95%) as acolorless oil.

¹H-NMR(CDCl₃, 250 MHz) δ5.14(s, 1H, H-5), 4.1(d, 1H, J=7.5 Hz, H-12),3.57–3.52(t, 2H, J=7.5 Hz), 2.31(s, 1H), 1.94–1.71(m, 6H), 1.54(s, 3H,CH₃-15), 1.53–1.25(m, 9H), 0.84(d, 6H, J=5.0 Hz, CH₃-13,14).

¹³C-NMR(CDCl₃, 63 MHz) δ135.5, 120.7, 69.8, 42.8, 42.4, 39.1, 38.9,37.8, 36.0, 31.9, 28.0, 27.0, 26.4, 26.1, 24.1, 20.1, 10.2.

IR(neat) u_(max) 3427(OH), 2910, 1726, 1447, 1378, 1259, 992, 908, 734cm⁻¹

MS(EI) m/z 328([M+]), 310([M+]—H₂O), 249([M+]—Br).

(3) Synthesis of the Title Compound

12-(2′-bromoethyl)dihydroartemisinyl alcohol (XV)(250 mg, 0.665 mmol)was added to CH₃CN/CH₂Cl₂ (1/1, 60 mL) containing catalytic Rose Bengaland then was irradiated with white light (500 W tungsten lamp) at −23°C. for 4 hours under oxygen. After completion of the reaction, to thereaction mixture was added saturated NaHCO₃ solution (50 mL). Thismixture was extracted with diethyl ether (20 mL×3) and then washed withbrine (20 mL×2). The extract was dried over MgSO₄. Solvent wasevaporated under reduced pressure and the residue was dissolved inCH₃CN/CH₂Cl₂ (9/1, 10 mL). This solution was cooled to −40° C. Acidiccatalyst trifluoroacetic acid (TFA) was added to this solution and thenwas allowed to stir at −40° C. for 12 hours under oxygen. The reactionmixture was quenched with saturated NH₄Cl solution (10 mL) and extractedwith diethyl ether (20 mL×3). The extract was washed with water (30mL×2) and brine (30 mL×2), dried over MgSO₄, concentrated in vacuo andpurified by silica gel column (hexane/ethyl acetate=5/1 as eluent) toafford 12-(2′-bromoethyl)deoxoartemisinin (V)(99 mg, 40%) as a whitesolid.

[α]¹⁸ _(D)=+96.3(c 0.1, CHCl₃).

m.p. 94° C.

¹H-NMR(CDCl₃, 500 MHz) δ5.26(s, 1H, H-5), 4.33–4.27(m, 1H, H-12),3.56–3.51(m, 2H, H-2′), 2.60–2.45(m, 2H), 2.30(ddd, 1H, J=4.1, 3.8, 4.1Hz), 2.05–1.89(m, 4H), 1.83–1.48(m, 4H), 1.39(s, 3H, CH₃-15),1.28–1.22(m, 2H), 0.94(d, 3H, J=4.8 Hz, CH₃-13), 0.87(d, 3H, J=7.4 Hz,CH₃-14), 0.78(m, 1H).

¹³C-NMR(CDCl₃, 125 MHz) δ103.4, 89.5, 81.2, 73.2, 52.4, 44.4, 37.7,37.3, 35.8, 34.8, 33.7, 31.6, 30.3, 26.3, 25.0, 20.4, 13.1.

IR(KBr) u_(max) 2950, 1451, 1377, 1272, 1117, 1042, 1010, 880(O—O), 756cm⁻¹

MS(EI) m/z 376(M+2), 342([M+]-O₂).

Example 2 Synthesis of 12-(2′-aminoethyl)deoxoartemisinin (VI) (1)Synthesis of 12-(2′-ethyl azide)deoxoartemisinin (XVI)

12-(2′-bromoethyl)deoxoartemisinin (V) (128 mg, 0.352 mmol) wasdissolved in DMF (5 mL) and sodium azide (45.7 mg, 0.704 mmol) wasadded. This mixture was allowed to stir at room temperature for 5 hours.To the reaction mixture was added water (30 mL). The resulting mixturewas extracted with ethyl acetate (50 mL×2) and then washed with brine(40 mL×2). The extract was dried over MgSO₄, concentrated in vacuo, thenpurified by silica gel column (hexane/ethyl acetate=5/2 as eluent) toafford 12-(2′-ethyl azide)deoxoartemisinin (XVI) (109 mg, 92%) as acolorless oil.

[α]²³ _(D)=+64.2(c 0.47, CHCl₃).

¹H-NMR (CDCl₃, 250 MHz) δ5.28(s, 1H, H-5), 4.31–4.27(m, 1H, H-12),3.56–3.53(m, 1H), 3.44–3.38(m, 1H), 2.69–2.64(m, 1H), 2.31(ddd, 1H,J=4.1, 3.8, 4.1 Hz), 2.03–1.75(m, 5H), 1.67–1.60(m, 3H), 1.40(s, 3H,CH₃-15), 1.33–1.27(m, 3H), 0.96(d, 3H, J=5.6 Hz, CH₃-13), 0.87(d, 3H,J=7.5 Hz, CH₃-14), 0.82(m, 1H).

¹³C-NMR (CDCl₃, 63 MHz) δ103.5, 89.5, 81.3, 72.3, 52.5, 49.8, 44.5,37.8, 36.9, 34.7, 30.4, 29.5, 26.3, 25.1, 25.1, 20.4, 13.1.

IR(neat) u_(max) 2927, 2875, 2095(N₃), 1733, 1454, 1377, 1277, 1098,1011, 880(O—O), 756 cm⁻¹

MS(EI) m/z 337[M+], 305([M+]-O₂).

(2) Synthesis of the Title Compound

12-(2′-ethylazide)deoxoartemisinin (XVI) (137.9 mg, 0.352 mmol) wasdissolved in dry THF (10 mL), cooled to −78° C. and LAH (35.1 mg, 0.925mmol) was added. The solution was allowed to stir at −78° C. for 1 hour,warmed to −10° C. slowly and allowed to stir at that temperature for 1hour. The reaction mixture was extracted with ethyl acetate (50 mL×2)and washed with brine (40 mL×2). The extract was dried over MgSO₄,concentrated in vacuo, then purified by silica gel column (100% methanolas eluent) to afford 12-(2′-aminoethyl)deoxoartemisinin (VI) (85.4 mg,78%) as a white solid.

[α]²³ _(D)=+38.7 (c 0.1, CHCl₃).

m.p. 103° C.

¹H-NMR (CDCl₃, 250 MHz) δ5.32(s, 1H, H-5), 4.29–4.21(m, 1H, H-12),2.93–2.84(m, 3H), 2.69–2.64(m, 1H), 2.32(ddd, 1H, J=4.0, 3.7, 4.0 Hz),2.05–1.82(m, 3H), 1.80–1.74(m, 2H), 1.62–1.50(m, 2H), 1.40(s, 3H,CH₃-15), 1.32–1.26(m, 4H), 0.96(d, 3H, J=5.7 Hz, CH₃-13), 0.87(d, 3H,J=7.5 Hz, CH₃-14), 0.83(m, 1H).

¹³C-NMR (CDCl₃, 63 MHz) δ103.5, 89.4, 81.5, 74.2, 52.7, 44.7, 41.0,37.8, 36.9, 34.8, 33.2, 30.6, 26.5, 25.1, 25.0, 20.5, 13.4.

IR(KBr) u_(max) 3365(NH), 2924, 2874, 1663, 1570, 1455, 1377, 1114,1054, 1011, 944, 877(O—O), 753 cm⁻¹

HRMS(FAB) m/z 312.2175([M+H]⁺, obsd), 311.2097(calcd for C₁₇H₂₉NO₄).

Elemental analysis (C₁₇H₂₉NO₄) C, H, N.

Example 3 Synthesis of 12-(2′-ethylsulfide)deoxoartemisinin Dimer (IVa₁)

12-(2′-bromoethyl)deoxoartemisinin (V) (45 mg, 0.124 mmol) was dissolvedin pure ethanol (4 mL), allowed to stir at room temperature for 10minutes and Na₂S (4.8 mg, 0.5 eq) was added. The reaction mixture wasallowed to stir at room temperature for 7 hours. This reaction mixturewas extracted with ethyl acetate (10 mL×3) and washed with brine (10mL×2). The extract was dried over MgSO₄, concentrated in vacuo, thenpurified by silica gel column (hexane/ethyl acetate=5/1 as eluent) toafford 12-(2′-ethylsulfide)deoxoartemisinin dimer (IVa₁) (58.7 mg, 76%)as a colorless oil.

[α]²⁵ _(D)=+58.7 (c 0.23, CHCl₃).

¹H-NMR (CDCl₃, 250 MHz) δ5.29(s, 2H, H-5), 4.24–4.19(m, 2H, H-12),2.91–2.84(m, 2H), 2.71–2.70(m, 2H), 2.55–2.49(m, 2H), 2.33(ddd, 2H,J=3.1, 3.5, 4.0 Hz), 2.03–1.78(m, 8H), 1.67–1.55(m, 10H), 1.41(s, 6H,CH₃-15), 1.36–1.26(m, 4H), 0.96(d, 6H, J=5.8 Hz, CH₃-13), 0.88(d, 6H,J=7.4 Hz, CH₃-14), 0.83(m, 2H).

¹³C-NMR (63 MHz, CDCl₃) δ103.6, 89.2, 81.4, 76.8, 75.5, 52.7, 44.8,37.7, 36.9, 34.8, 30.5, 30.1, 26.5, 25.2, 25.0, 20.6, 13.5.

IR(neat) u_(max) 2925, 2876, 1617, 1459, 1379, 1119, 1054, 1011,887(O—O), 735 cm⁻¹

HRMS(FAB) m/z 645.3541([M+Na]⁺, obsd), 622.3539(calcd for C₃₄H₅₄O₈S).

Elemental analysis (C₃₄H₅₄O₈S) C, H, S.

Example 4 Synthesis of 12-(2′-sulfonylethyl)deoxoartemisinin dimer(IVa₂)

12-(2′-ethylsulfide)deoxoartemisinin dimer (IVa₁) (28 mg, 0.041 mmol)was dissolved in dry CH₂Cl₂ (2 mL), allowed to stir at room temperaturefor 10 minutes and m-CPBA (15.7 mg, 0.091 mmol) was added slowly. Thereaction mixture was allowed to stir at room temperature for 3 h andsaturated NaHCO₃ solution (3 mL) was added. The reaction mixture wasextracted with ethyl acetate (10 mL×3) and washed with brine (10 mL×2).The extract was dried over MgSO₄, concentrated in vacuo, then purifiedby silica gel column (hexane/ethyl acetate=2/1 as eluent) to afford12-(2′-sulfonylethyl)deoxoartemisinin dimer (IVa₂) (24.6 mg, 91%) as awhite solid.

[α]²⁰ _(D)=+84.2 (c 0.44, CHCl₃).

m.p. 98° C.

¹H-NMR (CDCl₃, 250 MHz) δ5.30(s, 2H, H-5), 4.25–4.18(m, 2H, H-12),3.53–3.41(m, 2H, H-2), 3.05–2.935(m, 2H, H-2), 2.79–2.71(m, 2H),2.36(ddd, 2H, J=3.2, 3.5, 4.1 Hz), 2.17–1.92(m, 6H), 1.88–1.78(m, 4H),1.69–1.49(m, 8H), 1.39(s, 6H, CH₃-15), 0.97(d, 6H, J=5.7 Hz, CH₃-13),0.92(d, 6H, J=7.6 Hz, CH₃-14), 0.89(m, 2H).

¹³C NMR (CDCl₃, 63 MHz) δ134.1, 130.6, 130.1, 128.6, 103.6, 89.4, 81.4,74.1, 52.5, 44.4, 36.8, 30.5, 26.4, 25.2, 22.4, 20.4, 13.2.

IR(KBr) u_(max) 2928, 2876, 1723, 1575, 1449, 1380, 1280, 1123, 1052,880(O—O), 734 cm⁻¹

HRMS(FAB) m/z 677.3335([M+Na]⁺, obsd), 654.3438(calcd for C₃₄H₆₄O₁₀S).

Elemental analysis (C₃₄H₆₄O₁₀S) C, H, S.

Example 5 Synthesis of S,S′-[12-(2′-ethyl)deoxoartemisinin]dithiopropanedimer (IVb₁)

Powdered KOH (12.36 mg, 0.22 mmol) was added to DMSO (2 mL), allowed tostir at room temperature for 1 hour and then 1,3-propanedithiol (6.64μl, 0.054 mmol) and 12-(2′-bromoethyl)deoxoartemisinin (V) (41 mg, 0.112mmol) was added together. The reaction mixture was allowed to stir atroom temperature for 1 hour. The reaction mixture was extracted withethyl acetate (30 mL×3) and washed with brine (20 mL×2). The extract wasdried over MgSO₄, concentrated in vacuo, then purified by silica gelcolumn (hexane/ethyl acetate=5/2 as eluent) to affordS,S′-[12-(2′-ethyl)deoxoartemisinin]dithiopropane dimer (IVb₁) (50.7 mg,65%) as a colorless oil.

[α]²⁴ _(D)=+112.3(c 0.4, CHCl₃).

¹H-NMR (CDCl₃, 250 MHz) δ5.29(s, 2H, H-5), 4.22–4.17(m, 2H, H-12),2.83–2.66(m, 10H), 2.41–2.29(m, 2H), 2.05–1.76(m, 10H), 1.66–1.53(m,10H), 1.41(s, 6H, CH3–15), 1.33–1.26(m, 4H), 0.96(d, 6H, J=5.7 Hz,CH₃-13) 0.88(d, 3H, J=7.5 Hz, CH₃-14), 0.83(m, 2H).

¹³C-NMR(CDCl₃, 63 MHz) δ103.6, 89.3, 81.4, 75.3, 52.7, 44.7, 38.8, 37.8,36.9, 34.8, 32.2, 30.5, 30.2, 29.1, 26.5, 25.2, 25.0, 20.5, 13.4.

IR(neat) u_(max) 2928, 2873, 1655, 1719, 1452, 1378, 1215, 1120, 1036,877(O—O), 755 cm⁻¹

HRMS(FAB) m/z 719.3701([M+Na]⁺, obsd) 696.3730(calcd for C₃₇H₆₀O₈S₂).

Elemental analysis (C₃₇H₆₀O₈S₂) C, H, S.

Example 6 Synthesis ofS,S′-[12-(2′-ethyl)deoxoartemisinin]disulfonylpropane Dimer (IVb₂)

S,S′-[12-(2′-ethyl)deoxoartemisinin]disulfonylpropane dimer (IVb₂) (22.6mg, 72%) as a white solid was prepared by following the procedure ofExample 4, but replacing 12-(2′-ethylsulfide)deoxoartemisinin dimer(IVa₁) with S,S′-[12-(2′-ethyl)deoxoartemisinin]dithiopropane dimer(IVb₁) (29 mg, 0.041 mmol) and using 31.1 mg of m-CPBA (0.18 mmol).

[α]²⁵ _(D)=+110.4(c 0.47, CHCl₃).

m.p. 138° C.

¹H-NMR (CDCl₃, 250 MHz) δ5.30(s, 2H, H-5), 4.25–4.21(m, 2H, H-12),3.49–3.43(m, 2H), 3.26(t, 2H, J=7.1 Hz), 3.04–2.92(m, 2H), 2.74–2.62(m,2H), 2.49–2.43(m, 2H), 2.08–1.94(m, 8H), 1.89–1.75(m, 4H), 1.69–1.59(m,8H), 1.39(s, 6H, CH₃-15), 1.34–1.27(m, 4H), 0.97(d, 6H, J=5.7 Hz,CH₃-13), 0.91(d, 6H, J=7.6 Hz, CH₃-14), 0.88(m, 2H).

¹³C-NMR(CDCl₃, 63 MHz) δ134.2, 130.6, 130.1, 128.4, 103.6, 89.5, 81.4,74.1, 52.5, 44.3, 37.7, 36.8, 34.7, 31.3, 30.5, 26.4, 25.2, 20.4, 13.5.

IR(KBr) u_(max) 2926, 2875, 1720, 1584, 1495, 1387, 1310, 1130, 1052,877(O—O), 756, 465 cm⁻¹

HRMS(FAB) m/z 783.3538([M+Na]⁺, obsd), 760.3526(calcd for C₃₇H₆₀O₁₂S₂).

Elemental analysis (C₃₇H₆₀O₁₂S₂) C, H, S.

Example 7 Synthesis of 12-(2′-amidethyl)deoxoartemisinin Dimer (IVc)

12-carboxylethyldeoxoartemisinin (VII) (32 mg, 0.086 mmol), prepared bya known procedure (see Jung, M.; Freitas, A. C. C.; McChesney, J. D.;ElSohly, H. N., A Practical and General Synthesis of(+)-Carboxyalkyldeoxoartemisinins, Heterocycles. 1994, 39, 23–29), wasdissolved in dry CH₂Cl₂ (3 mL) and HOBt (38 mg, 0.256 mmol) and EDC (47mg, 0.256 mmol) was added together. The reaction mixture was allowed tostir at room temperature for 30 minutes and12-(2′-aminoethyl)deoxoartemisinin (VI) (30 mg, 0.096 mmol) was added.The resulting reaction mixture was allowed to stir at room temperaturefor 4 hours. The reaction mixture was extracted with ethyl acetate (20mL×3) and washed with brine (10 mL×2). The extract was dried over MgSO₄,concentrated in vacuo, then purified by silica gel column (hexane/ethylacetate=1/2 as eluent) to afford 12-(2′-aminoethyl)deoxoartemisinindimer (IVc) in 81% yield as a colorless oil.

[α]²³ _(D)=+111.3 (c 0.38, CHCl₃).

¹H-NMR (CDCl₃, 500 MHz) δ6.28(s, 1H, NH), 5.30(s, 1H, H-5), 5.29(s, 1H,H-5), 4.33–4.31(m, 1H, H-12), 4.06–4.04(m, 1H, H-12), 3.58–3.56(m, 1H,H-2), 3.28–3.26(m, 1H, H-2), 2.73–2.69(m, 1H), 2.65–2.62(m, 1H),2.51–2.44(m, 1H), 2.35–2.32(t, 2H, J=13.5 Hz), 2.24–2.15(m, 1H),2.04–1.98(m, 2H), 1.95–1.89(m, 3H), 1.78–1.71(m, 4H), 1.66–1.64(M, 4H),1.40(s, 6H, CH₃-15), 1.37–1.24(m, 9H), 0.96(d, 3H, J=5.3 Hz, CH₃-13),0.95(d, 3H, J=5.7 Hz, CH₃-13), 0.88(d, 3H, J=7.5 Hz, CH₃-14), 0.86(d,3H, J=7.5 Hz, CH₃-14), 0.84(m, 2H).

¹³C-NMR (CDCl₃, 63 MHz,) δ173.8, 126.8, 126.1, 118.0, 111.2, 103.7,103.5, 89.6, 89.0, 81.5, 81.4, 76.4, 74.7, 52.8, 52.5, 44.8, 44.3, 37.8,37.6, 36.8, 35.1, 34.8, 34.7, 30.7, 30.5, 26.5, 26.4, 25.4, 25.1, 25.0,20.5, 20.4, 13.7, 13.6.

IR(neat) u_(max) 3380(NH), 2941, 2877, 1653(C═O), 1545, 1446(C—N), 1379,1097, 1051, 1013, 915, 878(O—O), 733 cm⁻¹

HRMS(FAB) m/z 634.3995([M+H]⁺, obsd), 633.3877(calcd for C₃₅H₅₅NO₉).

Elemental analysis (C₃₅H₅₅NO₉) C, H, N.

Example 8 Synthesis of 12-[2′-(N-glutamic)-α,β-amide]deoxoartemisininDimer (IVd) (1) Synthesis of12-[2′-(N-tBOC-glutamic-γ-benzylester)-α-amide]deoxoartemisinin (VIII)

N-tBOC-L-glutamic acid-γ-benzylester (35 mg, 0.11 mmol) was dissolved indry CH₂Cl₂ (5 mL) and HOBt (52 mg, 0.342 mmol) and EDC (63 mg, 0.342mmol) was added. The reaction mixture was allowed to stir at roomtemperature for 30 minutes and 12-(2′-aminoethyl)deoxoartemisinin (VI)(42 mg, 0.135 mmol) was added. The resulting reaction mixture wasallowed to stir at room temperature for 3 hours. The reaction mixturewas extracted with ethyl acetate (20 mL×3) and washed with brine (10mL×2). The extract was dried over MgSO₄, concentrated in vacuo, thenpurified by silica gel column (hexane/ethyl acetate=1/2 as eluent) toafford 12-[N-tBOC-glutamic-γ-benzylester)-α-amide]deoxoartemisinin(VIII) (71.5 mg, 84%) as a colorless oil.

[α]²⁵ _(D)=+73.6 (c 0.47, CHCl₃).

¹H-NMR (CDCl₃, 250 MHz) δ7.36(s, 5H, aromatic H), 7.02(br, 1H, NH),5.33(s, 1H, H-5), 5.11(s, 2H, benzyl), 4.40–4.39(m, 1H, H-12),4.29–4.27(m, 1H), 4.15–4.13(m, 1H), 3.58–3.56(m, 1H, H-2), 3.28–3.24(m,1H, H-2), 2.61–2.43(m, 3H), 2.36–2.14(m, 3H), 2.12–1.98(m, 4H),1.91–1.65(m, 4H), 1.44(s, 9H, t-BOC), 1.42(s, 3H, CH₃-15), 1.35–1.26(m,3H), 0.96(d, 3H, J=5.6 Hz, CH₃-13), 0.85(d, 3H, J=7.6 Hz, CH₃-14),0.82(m, 1H).

¹³C-NMR (CDCl₃, 63 MHz) δ173.2, 171.5, 155.2, 136.1, 128.8, 128.8,128.5, 128.5, 128.4, 128.4, 103.8, 89.9, 81.3, 74.4, 66.6, 61.2, 53.1,52.3, 44.0, 39.4, 37.7, 36.7, 34.6, 30.8, 28.6, 28.5, 28.5, 26.2, 25.1,25.0, 20.4, 12.6.

IR(neat) u_(max) 3364(NH), 2932, 2876, 1736(C═O), 1663(C═O), 1538, 1453,1393, 1249, 1163, 1051, 880(O—O), 702, 610 cm⁻¹

HRMS(FAB) m/z 631.3507([M+H]⁺, obsd), 630.3516(calcd for C₃₄H₅₀N₂O₉).

(2) Synthesis of 12-[2′-(N-tBOC-glutamic acid)-α-amide]deoxoartemisinin(IX)

12-[2′-(N-tBOC-glutamic-γ-benzylester)-α-amide]deoxoartemisinin (VIII)(36 mg, 0.057 mmol) was dissolved in dry THF/H₂O (1/1, 5 mL) and 1N LiOH(1 mL) was added. The solution was allowed to stir at room temperaturefor 2 hours. The reaction solution was acidified with 1N HCl, extractedwith ethyl acetate (20 mL×3), and then washed with brine (10 mL×2). Theextract was dried over MgSO₄, concentrated in vacuo, then purified bysilica gel column (hexane/ethyl acetate=1/2 as eluent) to afford12-[2′-(N-tBOC-glutamic acid)-α-amide]deoxoartemisinin (IX) (26.8 mg,87%) as a colorless oil.

[α]²⁵ _(D)=+66.4 (c 0.34, CHCl₃).

¹H—NMR(CDCl₃, 250 MHz) δ5.60(d, 1H, J=7.9 Hz, NH), 5.33(s, 1H, H-5),4.34–4.32(m, 1H, H-12), 4.20–4.15(m, 1H), 3.49–3.47(m, 1H, H-2),3.33–3.31(m, 1H, H-2), 2.66–2.58(m, 1H), 2.48–2.40(m, 2H), 2.30(ddd, 1H,J=2.2, 1.9, 3.7 Hz), 2.03–1.73(m, 5H), 1.67–1.61(m, 6H), 1.41(s, 9H,t-BOC), 1.38(s, 3H, CH₃-15), 1.33–1.21(m, 3H), 0.95(d, 3H, J=5.1 Hz,CH₃-13), 0.84(d, 3H, J=7.4 Hz, CH₃-14), 0.81(m, 1H).

¹³C-NMR ((CDCl₃, 63 MHz,) δ176.6, 172.2, 103.7, 89.7, 81.4, 76.9, 74.7,53.4, 52.5, 44.3, 39.1, 37.8, 36.8, 34.7, 30.7, 30.6, 29.8, 28.7, 28.7,28.7, 28.7, 26.2, 25.2, 25.0, 20.5, 13.0.

IR(neat) u_(max) 3352(CO₂H and NH), 2933, 2879, 1714(C═O), 1657(C═O),1533, 1459, 1379, 1275, 1170, 1053, 914, 882(O—O), 733 cm⁻¹

LCMS(ESI) m/z 540([M+]).

(3) Synthesis of 12-[2′-(N-tBOC-glutamic)-α,β-amide]deoxoartemisininDimer (X)

12-[2′-(N-tBOC-glutamic acid)-α-amide]deoxoartemisinin (IX) (21 mg,0.039 mmol) was dissolved in dry CH₂Cl₂ (2 mL) and HOBt (22 mg, 0.119mmol) and EDC (29 mg, 0.119 mmol) were added together. The reactionmixture was allowed to stir at room temperature for 30 minutes and12-(2′-aminoethyl)deoxoartemisinin (VI) (18 mg, 0.058 mmol) was added.The resulting reaction mixture was allowed to stir at room temperaturefor 3 hours. The reaction mixture was extracted with ethyl acetate (20mL×3) and washed with brine (10 mL×2). The extract was dried over MgSO₄,concentrated in vacuo, then purified by silica gel column (hexane/ethylacetate=1/2 as eluent) to afford12-[2′-(N-tBOC-glutamic)-α,β-amide]deoxoartemisinin dimer (X)(16.6 mg,51%) as a colorless oil.

[α]²⁵ _(D)=+114.6(c 0.46, CHCl₃).

¹H-NMR (CDCl₃, 250 MHz) δ6.71(s, 1H, NH), 5.87(s, 1H, NH), 5.34(s, 1H,H-5) 1H, H-5), 4.34–4.31(m, 2H, H-12), 4.13–4.10(m, 1H), 3.53–3.48(m,2H), 3.25–3.23(m, 2H), 2.62–2.60(m, 2H), 2.39–2.29(m, 6H), 2.05–1.88(m,8H), 1.86–1.62(m, 12H), 1.43(s, 9H, t-BOC), 1.39(s, 6H, CH₃-15),1.28–1.25(m, 5H), 0.96(d, 6H, J=5.3 Hz, CH₃-13), 0.86(d, 6H, J=7.5 Hz,CH₃-14), 0.83(m, 2H).

¹³C-NMR (CDCl₃, 63 MHz,) δ173.3, 173.2, 171.5, 171.4, 103.5, 103.4,89.8, 89.6, 81.4, 81.4, 76.9, 74.3, 52.5, 52.5, 44.4, 44.3, 39.3, 37.8,36.8, 34.7, 30.8, 28.9, 28.7, 26.4, 25.2, 25.1, 20.5, 14.5, 13.0, 12.8.

IR(neat) u_(max) 3379(NH), 2877, 1713(C═O), 1656(C═O), 1545, 1451, 1379,1268, 1030, 917, 880(O—O), 732 cm⁻¹

LCMS(ESI) m/z 834([M+H]).

(4) Synthesis of the Title Compound

12-[2′-(N-tBOC-glutamic)-α,β-amide]deoxoartemisinin dimer (X) (28 mg,0.036 mmol) was dissolved in dry CH₂Cl₂ (2 mL). To this solution wasslowly added TFA (4.92 mg, 1.2 eq) at 0° C. for 30 minutes. Theresulting reaction mixture was allowed to stir at 0° C. for 4 hours,extracted with ethyl acetate (20 mL×3) and washed with brine (10 mL×2).The extract was dried over MgSO₄, concentrated in vacuo, then purifiedby silica gel column (hexane/ethyl acetate=1/2 as eluent) to afford12-[2′-(N-glutamic)-α,β-amide]deoxoartemisinin dimer (IVd) (19 mg, 72%)as a colorless oil. The dimer (IVd) is five times more water soluble(5.21 mg/ml) than artemisinin.

[α]²⁵ _(D)=+120.8 (c 0.48, CHCl₃).

¹H-NMR (CDCl₃, 250 MHz) δ6.83(s, 1H, NH), 5.74(s, 1H, NH), 5.34(s, 1H,h-5), 5.31(s, 1H, H-5), 4.35–4.33(m, 2H, H-12), 4.15–4.12(m, 1H),3.53–3.49(m, 2H), 3.27–3.24(m, 2H), 2.64–2.62(m, 2H), 2.32–2.30(m, 2H),2.15–1.58(m, 12H), 1.53–1.41(m, 10H), 1.39(s, 6H, CH₃-15), 1.34–1.28(m,6H), 0.97(d, 6H, J=5.3 Hz, CH₃-14), 0.86(d, 6H, J=7.5 Hz, CH₃-14),0.82(m, 2H).

¹³C-NMR (63 MHz, CDCl₃) δ173.4, 173.3, 171.5, 171.3, 103.6, 103.4, 89.8,89.7, 84.4, 84.3, 76.9, 74.3, 52.6, 52.4, 44.5, 44.3, 39.3, 37.9, 36.7,34.6, 30.9, 30.8, 28.7, 26.4, 25.2, 25.1, 20.5, 20.4, 14.5, 13.3, 12.7.

IR(neat) u_(max) 3367(NH), 3098(NH), 2956, 2868, 1689(C═O), 1558, 1446,1380, 1209, 1137, 998, 887(O—O), 847, 757, 729 cm⁻¹

HRMS(FAB) m/z 734.4592([M+H]⁺, obsd), 733.4513(calc for C₃₉H₆₃N₃O₁₀).

Elemental analysis (C₃₉H₆₃N₃O₁₀) C, H, N.

Example 9 Synthesis of Deoxoartemisinin Trimer (IVe) (1) Synthesis ofN-[12-(β-deoxoartemisinin)propionyl]-L-glutamic diethyl ester (XI)

12-carboxylethyldeoxoartemisinin (VII)(32 mg, 0.256 mmol) was dissolvedin dry CH₂Cl₂ (3 mL) and HOBt (38 mg, 0.256 mmol) and EDC (47 mg, 0.256mmol) were added together. The reaction mixture was allowed to stir atroom temperature for 30 minutes and L-glutamic diethylester (36 mg,0.171 mmol) was added. The resulting reaction mixture was allowed tostir at room temperature for 3 hours. The reaction mixture was extractedwith ethyl acetate (20 mL×3) and washed with brine (10 mL×2). Theextract was dried over MgSO₄, concentrated in vacuo, then purified bysilica gel column (hexane/ethyl acetate=1/2 as eluent) to affordN-[12-(β-deoxoartemisinin)propionyl]-L-glutamic diethylester (XI) (37.9mg, 84%) as a colorless oil.

[α]²⁵ _(D)=+44.5 (c 0.47, CHCl₃).

¹H-NMR (CDCl₃, 250 MHz,) δ6.31(d, 1H, J=7.5 Hz, NH), 5.29(s, 1H, H-5),4.63–4.56(m, 1H), 4.23–4.08(m, 5H), 2.75–2.71(m, 1H), 2.50–2.18(m, 7H),2.04–1.65(m, 9H), 1.40(s, 3H, CH₃-15), 1.31(s, 3H), 1.28(s, 3H),1.22–1.15(m, 2H), 0.96(d, 3H, J=5.8 Hz, CH₃-13), 0.88(d, 3H, J=7.5 Hz,CH₃-14), 0.85(m, 1H).

¹³C-NMR (CDCl₃, 63 MHz) δ173.2, 172.3, 103.7, 89.1, 81.5, 76.8, 76.3,61.9, 61.0, 52.7, 52.0, 44.8, 37.7, 36.8, 34.8, 34.8, 31.2, 30.7, 30.5,27.8, 26.4, 25.2, 25.0, 20.5, 14.5, 13.5.

IR(neat) u_(max) 3370(NH), 2939, 2878, 1737(C═O), 1669(C═O), 1533, 1446,1372, 1054, 1012, 880(O—O), 742 cm⁻¹

MS(FAB) m/z 526.5([M+H]⁺).

Elemental analysis (C₂₇H₄₃NO₉) C, H, N.

(2) Synthesis of N-[12-(3-deoxoartemisinin)propionyl]-L-glutamic diacid(XII)

N-[12-(β-deoxoartemisinin)propionyl]-L-glutamic diethylester (XI) (2 mg,0.08 mmol) was dissolved in TBF/H₂O (1/1, 5 mL), 1N LiOH (1 mL) wasadded and then was allowed to stir at room temperature for 2 hours. Tothe reaction mixture was added 1N HCl (1 mL). The resulting reactionmixture was extracted with ethyl acetate (20 mL×3) and washed with brine(10 mL×2). The extract was dried over MgSO₄, concentrated in vacuo, thenpurified by silica gel column (hexane/ethyl acetate=1/2 as eluent) toafford N-[12-(β-deoxoartemisinin)propionyl]-L-glutamic diacid (XII)(30.8 mg, 82%) as a colorless oil.

[α]²⁶ _(D)=+76.8 (c 0.22, CHCl₃).

m.p. 106° C.

¹H-NMR (CDCl₃, 250 MHz) δ8.96(br, 2H, CO₂H), 7.08(d, 1H, 3=6.5 Hz, NH),5.34(s, 1H, H-5), 4.62–4.60(m, 1H), 4.12–4.04(m, 1H, H-12), 2.72–2.70(m,1H), 2.48–2.26(m, 6H), 2.08–1.98(m, 3H), 1.82–1.78(m, 3H), 1.71–1.42(m,4H), 1.41(s, 3H, CH₃-14), 1.27–1.21(m, 2H), 0.95(d, 3H, J=5.3 Hz,CH₃-13), 0.87(d, 3H, J=7.2 Hz, CH₃-14), 0.84(m, 1H).

¹³C-NMR (CDCl₃, 63 MHz) δ177.3, 176.6, 175.1, 174.7, 104.3, 89.0, 81.5,76.9, 52.8, 52.1, 44.9, 37.6 36.8, 34.8, 34.5, 30.4, 27.1, 26.2, 25.1,24.9, 21.0, 20.6, 13.7.

IR(KBr) u_(max) 3346(CO₂H), 2942, 2877, 1728(C═O), 1631(C═O), 1539,1453, 1381, 1202, 1051, 912, 880(O—O), 732 cm⁻¹

LCMS(ESI) m/z 492 ([M+Na]⁺).

Elemental analysis (C₂₃H₃₅NO₉) C, H, N.

(3) Synthesis of the Title Compound

N-[12-(β-deoxoartemisinin)propionyl]-L-glutamic diacid (XII) (22 mg,0.047 mmol) was dissolved in dry CH₂Cl₂ (2 mL) and HOBt (27 mg, 0.141mmol) and EDC (31 mg, 0.141 mmol) were added. The reaction mixture wasallowed to stir at room temperature for 30 minutes and12-(2′-aminoethyl)deoxoartemisinin (VI) (29 mg, 0.093 mmol) was added.The resulting reaction mixture was allowed to stir at room temperaturefor 19 hours. The reaction mixture was extracted with ethyl acetate (30mL×3) and washed with brine (20 mL×2). The extract was dried over MgSO₄,concentrated in vacuo, then purified by silica gel column (hexane/ethylacetate=1/2 as eluent) to afford deoxoartemisinin trimer (IVe) (73.3 mg,74%) as a colorless solid.

[α]²⁴ _(D)=+102.7 (c 0.41, CHCl₃).

m.p. 138° C.

¹H-NMR (CDCl₃, 250 MHz) δ7.13–7.04(m, 2H, NH), 6.83–6.81(m, 1H, NH),5.32(s, 2H, H-5), 5.29(s, 1H, H-5), 4.39–4.34(m, 3H, H-12), 4.29–4.26(m,1H), 3.55–3.48(m, 2H), 3.32–3.27(m, 2H), 2.65–2.63(m, 3H), 2.36–2.31(m,7H), 2.17–1.93(m, 11H), 1.91–1.51(m, 17H), 1.39(s, 9H, CH₃-15),1.33–1.21(m, 7H), 0.95(d, 9H, J=5.4 Hz, CH₃-13), 0.88–0.83(m, 9H,CH₃-14), 0.82(m, 3H).

¹³C-NMR (CDCl₃, 63 MHz) δ174.4, 174.3, 173.2, 173.1, 103.5, 103.4, 89.9,89.8, 81.5, 81.4, 76.8, 53.2, 53.1, 52.8, 52.6, 52.4, 37.7, 36.8, 34.7,31.3, 30.8, 30.7, 30.0, 29.1, 26.4, 26.4, 25.1, 20.5, 20.4, 13.2, 13.1.

IR (KBr) u_(max) 3308(NH), 2933, 2875, 1667(C═O), 1535, 1465, 1377,1102, 1061, 1014, 938, 874(O—O), 751 cm⁻¹

HRMS (FAB) m/z 1056.6392([M+H]⁺, obsd), 1055.6294(calcd forC₅₇H₈₉N₃O₁₅).

Elemental analysis (C₅₇H₈₉N₃O₁₅) C, H, N.

Example 10 Synthesis of 12-(4′-aminobutyl)deoxoartemisinin (VI′) (1)Synthesis of 12-(4′-butyl azide)deoxoartemisinin

12-(4′-butyl azide)deoxoartemisinin (116.9 mg, 92%) was prepared as acolorless oil by following the procedure of Example 2 (1), but replacing12-(2′-bromoethyl)deoxoartemisinin with12-(4′-bromobutyl)deoxoartemisinin (137.9 mg, 0.352 mmol).

[α]²⁴ _(D)=+71.3 (c 0.1, CHCl₃).

¹H-NMR (CDCl₃, 250 MHz) δ5.29(s, 1H, H-5), 4.19–4.12(m, 1H, H-12),3.26(t, 2H, J=6.6 Hz, H-4), 2.64–2.62(m, 1H), 2.31(ddd, 1H, J=4.1, 3.8,4.1 Hz), 2.04–1.89(m, 2H), 1.68–1.65(m, 2H), 1.63–1.58(m, 7H), 1.40(s,3H, CH₃-15), 1.34–1.22(m, 2H), J=5.7 Hz, CH₃-13), 0.86(d, 3H, J=7.5 Hz),0.82(m, 1H).

¹³C-NMR (CDCl₃, 63 MHz), δ103.4, 89.4, 81.4, 75.3, 52.6, 51.7, 44.6,37.7, 36.9, 34.7, 30.6, 29.3, 29.0, 26.4, 25.2, 25.0, 25.0, 20.5, 13.2.

IR (neat) u_(max) 2927, 2876, 2095(N₃), 1597, 1454, 1379, 1255, 1097,1012, 946, 881(O—O), 643 cm⁻¹

MS (FAB) 366.4([M+H]⁺).

(2) Synthesis of the Title Compound

12-(4′-aminobutyl)deoxoartemisinin (VI′) (94.3 mg, 79%) was prepared asa white solid by following the procedure of Example 2 (2), but replacing12-(2′-ethyl azide)deoxoartemisinin with 12-(4′-butylazide)deoxoartemisinin (128.8 mg, 0.352 mmol).

[α]²⁵ _(D)=+49.4 (c 0.1, CHCl₃).

m.p. 105° C.

¹H-NMR (CDCl₃, 250 MHz) δ5.29(s, 1H, H-5), 4.16–4.09(m, 1H, H-12),2.71–2.66(m, 3H), 2.32(ddd, 1H, J=4.1, 3.8, 4.1 Hz), 2.08–2.04(m, 2H),1.87–1.72(m, 3H), 1.66–1.53(m, 4H), 1.51–1.45(m, 4H), 1.41(s, 3H,CH₃-15), 1.36–1.24(m, 4H), 0.96(d, 3H, J=4.3 Hz), 0.86(d, 3H, J=7.5 Hz),0.83(m, 1H).

¹³C-NMR (CDCl₃, 63 MHz), δ103.5, 89.4, 81.5, 76.0, 52.6, 51.8, 44.8,38.4, 36.9, 34.8, 31.2, 29.4, 29.0, 26.5, 25.3, 25.2, 25.1, 20.5, 13.3.

IR(KBr) u_(max) 3378(NH), 2925, 1591, 1454, 1379, 1117, 1038, 1005,887(O—O), 748 cm⁻¹

HRMS(FAB) m/z 340.2402([M+H]⁺, obsd), 339.2410(calcd for C₁₉H₃₃NO₄).

Elemental analysis (C₁₉H₃₃NO₄) C, H, N.

Example 11 Synthesis of 12-(4′-aminobutyl)deoxoartemisinin dimer (IVc′)

12-(4′-aminobutyl)deoxoartemisinin dimer (IVc′) (46 mg, 81%) wasprepared as a colorless oil by following the procedure of Example 7, butreplacing 12-(2′-aminoethyl)deoxoartemisinin with12-(4′-aminobutyl)deoxoartemisinin (28 mg, 0.096 mmol).

[α]²³ _(D)=+104.2 (c 0.23, CHCl₃).

¹H-NMR (CDCl₃, 250 MHz) δ5.68(s, 1H, NH), 5.28(s, 2H, H-5), 4.13–4.03(m,2H, H-12), 3.25–3.21(m, 2H, H-4), 2.72–2.65(m, 2H), 2.39–2.21(m, 4H),2.11–1.67(m, 9H), 1.61–1.46(m, 10H), 1.40(s, 6H, CH₃-15), 1.36–1.22(m,7H), 0.96(d, 6H, J=4.6 Hz, CH₃-13), 0.89(d, 3H, J=7.4 Hz, CH₃-14),0.86(d, 3H, J=7.4 Hz, CH₃-14), 0.83(m, 2H).

¹³C-NMR (CDCl₃, 63 MHz) δ173.3, 126.5, 126.3, 118.2, 112.5, 103.7,103.6, 89.4, 89.1, 81.5, 76.9, 76.4, 75.9, 52.8, 52.5, 44.8, 44.3, 37.8,37.0, 36.4, 36.1, 35.2, 35.1, 34.8, 34.3, 30.6, 30.6, 26.6, 26.4, 25.2,25.1, 25.1, 25.1, 20.6, 20.5, 13.7, 13.6.

IR(neat) u_(max) 3388(NH), 2936, 2875, 1650(C═O), 1539, 1452(C—N), 1379,1216, 1097, 1051, 1005, 873(O—O), 753 cm⁻¹

HRMS(FAB) m/z 662.4173([M+H]⁺, obsd), 661.4190(calcd for C₃₇H₅₉NO₉).

Elemental analysis (C₃₇H₅₉NO₉) C, H, N.

Example 12 Measurement of Anticancer Activity

The anticancer activities of the deoxoartemisinin dimer and trimer ofthe present invention were measured by a known micro-culture tetrazoliumassay (see Carmichel, J.; DeGraff, W. G; Gazdar, A. F.; Minna, J. D.;Mitchell, J. B., Evaluation of a Tetrazolium-based SemiautomatedColorimetric Assay: Assessment of Chemosensitivity testing, Cancer Res.,1987, 47, 936–42).

The table 1 shows cytotoxicities of the compounds of the presentinvention on mouse and human cancer cells in vitro.

TABLE 1 In vitro cytotoxicities (IC₅₀ (μg/mL)) P388 EL4 Bewo HT-29PANC-1 SKOV3 MCF7 A549 Compound (IVa₁) 0.40 0.23 14.20 0.24 0.017Compound (IVa₂) 5.60 0.54 1.04 0.38 0.025 Compound (IVb) 8.40 10.00 8.500.38 5.6 Compound (IVc) 10.40 6.29 7.50 0.69 12.6 0.005 Compound(IVc′) >20 >20 >20 20.20 >20 Compound (IVd) 15.60 16.50 >20 6.50 15.3Compound (IVe) 0.12 1.07 18.30 0.09 2.69 11.2 0.017 2.45 Adriamycin 0.390.67 6.24 0.10 0.12 Mitomycin 1.50 3.94 0.85 0.02 0.93 1.85 Taxol 2.271.34 7.39 0.01 5.76 12.30 0.0001 P388: mouse fibroblast leukemia cellEL4: mouse thymoma cell Bewo: human choriocarcinoma cell HT-29: humancolorectal adenocarcinoma cell PANC-1: human pancreatic cancer cellSKOV3: human ovarian carcinoma cell MCF7: human breast carcinoma cellA549: human lung cancer cell

As shown in Table 1, the sulfide dimer (IVa₁) of the present inventionexhibited an anticancer activity on the mouse fibroblast leukemia cell(P388), said activity being comparable to that of adriamycin and beingat least 4-fold higher than that of mitomycin. Also, the trimer (IVe) ofthe present invention exhibited an anticancer activity on P388, saidactivity being at least 3-fold higher than that of adrimycin, at least12-fold higher than that of mitomycin and at least 20-fold higher thanthat of taxol. The sulfide dimer (IVa₁), sulfone dimer (IVa₂) and trimer(IVe) of the present invention exhibited an anticancer activity on themouse thymoma cell (EL4), said activity being comparable to that ofadriamycin. Most compounds had little anticancer activity on humanchoriocarcinoma cell (Bewo) but the sulfone dimer (IVa₂) had ananticancer activity on Bewo, which was comparable to that of mitomycinand was at least 6-fold higher than that of taxol and adriamycin. Thetrimer (IVe) of the present invention exhibited an anticancer activityon human colorectal adenocarcinoma cell (HT-29), said activity beingcomparable to that of adriamycin, and also exhibited an anticanceractivity on human pancreatic cancer cell, said activity being at least2-fold higher than that of taxol. Most compounds had little anticanceractivity on human ovarian carcinoma cell (SKOV3), while the amide dimer(IVc) and trimer (Ive) of the present invention had an anticanceractivity similar to that of taxol. The amide dimer (IVc), sulfide dimer(IVa₁), sulfone dimer (IVa₂) and trimer (IVe) had higher anticanceractivity on human breast carcinoma cell (MCF7). Especially, the amidedimer (IVc) of the present invention exhibited an anticancer activity onMCF7, said activity being at least 24-fold higher than that ofadriamycin and at least 200-fold higher than that of mitomycin. Most ofthe compounds had less anticancer activity on human lung cancer cell(A549), while the trimer (IVe) of the present invention had ananticancer activity on A549 similar to that of mitomycin.

From the results, it can be seen that the trimer (IVe) of the presentinvention has a very excellent anticancer activity on most mouse andhuman cancer cells. Also, it can be seen that the anticancer activity ofthe dimer of the present invention depends on the length of the linkerlocated between two deoxoartemisinins. In other words, the compounds(IVa₁), (IVa₂) and (IVc), in which a linker has one amide- or onesulfur-centered two ethylene groups, have superior anticancer activityto the compounds (IVa₁), (IVa₂) and (IVc), in which a linker is longerthan the length of two ethylene groups.

INDUSTRIAL APPLICABILITY

The deoxoartemisinin dimer and trimer of the present invention are C-12non-acetal-type and do not have linker containing C—O bonds, aromatic orunsaturated groups at C-12 positions, and thus are acid stable, lesstoxic and have higher anticancer activity.

Also, according to the preparation of the present invention, thedeoxoartemisinin analog is easily prepared in high yield.

1. A deoxoartemisinin compound of the

wherein Y is


2. A method for preparing deoxoartemisinin trimer of the followingformula, said method comprising the steps of: (a) coupling12-carboxylethyldeoxoartemisinin with L-glutamic diethylester; (b)hydrolyzing two ester groups of the product from said step (a); and (c)doubly coupling the product from said step (b) with 2 moles of12-aminoethyldeoxoartemisinin:


3. The method as claimed in claim 2, wherein said coupling reaction iscarried in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimideand 10-hydroxybenzotriazole (EDC/HOBt).
 4. A method for preparing of thefollowing formula, said method comprising the steps of: (a)hydroborative oxidizing a terminal olefin of 12-vinyldihydroartemisinylalcohol; (b) brominating the product from said step (a) with CBr₄/PPh₃;(c) photooxygenative cyclizing the product from said step (b); (d)reacting the product from said step (c) with sodium azide; and (e)reducing an azide group of the product from said step (d):